Study of the Cryptosporidium parvum DHFR-TS in the model system Saccharomyces cerevisiae

Abstract:

Cryptosporidium parvum is a ubiquitous protozoan that causes severe gastrointestinal disease. The severe diarrhea that results can be life-threatening, particularly for the very young, old, and immune-compromised. Currently there is no approved therapy for cryptosporidiosis. We have developed a method using the model organism Saccharomyces cerevisiae to rapidly screen for compounds effective against the C. parvum dihydrofolate reductase (DHFR) enzyme. The DHFR enzyme is essential and an effective drug target in other organisms. The yeast screening method has identified several compounds that are effective inhibitors of the C. parvum DHFR but also inhibit the human enzyme. Three related compounds have been identified in the yeast system that selectively inhibit the protozoal DHFR and not the human enzyme but are not extremely effective.The yeast system was also used to investigate the functional differences between the native C. parvum bifunctional DHFR-TS (dihydrofolate reductase-thymidylate synthase) enzyme and an engineered monofunctional DHFR domain alone. Yeast reliant on the DHFR-TS form show the expected synergy between trimetrexate, a DHFR inhibitor, and sulfanilamide, an inhibitor of dihydropteroate synthase (DHPS), another enzyme in the folate pathway. Yeast reliant on the C. parvum DHFR domain alone, however, did not show synergy between the two drugs. In vitro assays on purified enzyme revealed that the DHFR domain alone has a lower affinity than the DHFR-TS for trimetrexate and for the cofactor, NADPH. The DHFR domain alone also is much less stable and is a less efficient enzyme than its bifunctional counterpart.